Crystal forms of beta-nicotinamide mononucleotide

ABSTRACT

The invention relates to crystalline forms of a β-nicotinamide mononucleotide, methods of their preparation, and related pharmaceutical preparations thereof. The invention also relates to preparations suitable for nutraceutical, veterinary, and agriculturally-relevant uses.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 62/236,657, filed Oct. 2, 2015, which ishereby incorporated by reference herein in its entirety.

BACKGROUND

β-Nicotinamide mononucleotide (NMN) has recently gamed attention for itsuse in the treatment, amelioration, mitigation, slowing, arrest,prevention and/or reversal of age-associated degenerative changes, suchas age-related obesity, age-related increases in blood lipid levels,age-related decreases in insulin sensitivity, age-related decreases inmemory function, and age-related changes in eye function such as maculardegeneration.

Given the therapeutic benefits associated with this compound, there is aneed for improved compositions of NMN. Further, there is a need forimproved methods for preparing and formulating β-nicotinamidemononucleotide.

SUMMARY OF INVENTION

One aspect of the invention relates to a crystalline compound having thestructure of formula (I),

Another aspect of the invention relates to methods for preparing thecrystalline compounds of formula (I).

In certain embodiments, the present invention provides a pharmaceuticalpreparation suitable for use in a human patient, comprising acrystalline compound of formula (I), and one or more pharmaceuticallyacceptable excipients. In certain embodiments, the pharmaceuticalpreparations may be for use in treating or preventing a condition ordisease as described herein. In certain embodiments, the pharmaceuticalpreparations have a low enough pyrogen activity to be suitable forintravenous use in a human patient.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the XRPD patterns of β-nicotinamide mononucleotide (NMN)forms 1 and 2.

FIG. 2 shows the differential scanning calorimetry thermogram of Form 1.

FIG. 3 shows the differential scanning calorimetry thermogram of Form 2.

FIG. 4 shows ¹H NMR spectra of the amorphous NMN, NMN Form 1, and NMNForm 2 after drying under vacuum. As shown in the spectra, Form 1 issubstantially anhydrous, and Form 2 has about 1.1-1.2 DMSO molecules permolecule NMN.

FIG. 5 shows a comparison of XRPD patterns of amorphous NMN, amorphousNMN post-storage, and NMN Form 1.

FIG. 6 shows a dynamic vapor sorption isotherm for amorphous NMN.

FIG. 7 shows a dynamic vapor sorption change in mass plot for amorphousNMN. When a sample of amorphous NMN is exposed to atmospheric humidity,the sample goes through phases. FIG. 7 shows the mass change over time.The amorphous sample is hygroscopic until it deliquesces and startscrystallizing. The weight loss below 1000 min shows a crystallisationevent. Once crystallised, the material remains crystalline, retainingthe same XRPD pattern after a double cycle, but still shows the abilityto pick up mass reversibly (up to 9% w/w change).

FIG. 8 is an image showing single crystals of NMN Form 1, observed undera polarized microscope.

DETAILED DESCRIPTION OF THE INVENTION

In certain embodiments, the invention provides a crystalline compoundhaving the structure of formula (I),

In certain embodiments, a crystalline compound of formula (I) is notsolvated (e.g., the crystal lattice does not comprise molecules of asolvent). In certain embodiments, the crystalline compound of formula(I) is anhydrous, or substantially anhydrous. In certain alternativeembodiments, a crystalline compound of formula (I) is solvated. Incertain such embodiments, the crystalline compound of formula (I) is adimethylsulfoxide (DMSO) solvate.

Any crystalline compound described herein may be used in the manufactureof a medicament for the treatment of any diseases or conditionsdisclosed herein.

In certain embodiments, the compounds of the present invention canassemble into more than one crystal formation. In an exemplaryembodiment, the crystalline compound having the structure of formula (I)exists as “form I” and “form II”, as described in detail below. Thesedifferent forms are understood as “polymorphs” herein.

In certain embodiments, the polymorph of the crystalline compound ischaracterized by powder X-ray diffraction (XRD). θ represents thediffraction angle, measured in degrees. In certain embodiments, thediffractometer used in XRD measures the diffraction angle as two timesthe diffraction angle θ. Thus, in certain embodiments, the diffractionpatterns described herein refer to X-ray intensity measured againstangle 2θ.

In certain embodiments, an anhydrous crystalline compound of formula (I)has 2θ values 20.03; 20.14; 21.83; and 25.73. In further embodiments,the anhydrous crystalline compound has 2θ values 20.03; 20.14; 21.03;21.83; 23.08; 23.39; 25.73; and 26.59. In yet further embodiments, theanhydrous crystalline compound has 2θ values 7.70; 11.54; 12.64; 16.03;18.99; 20.03; 20.14; 20.83; 21.03; 21.83; 23.08; 23.39; 25.48; 25.73;26.59; and 29.78. In still yet further embodiments, the anhydrouscrystalline compound has 2θ values 7.70; 9.95; 11.54; 12.64; 16.03;18.18; 18.99; 19.16; 19.44; 20.03; 20.14; 20.83; 21.03; 21.83; 22.44;23.08; 23.39; 23.89; 24.08; 24.53; 24.68; 25.05; 25.48; 25.73; 26.08;26.59; 27.33; 27.67; 29.78; and 29.92.

In certain embodiments, an anhydrous crystalline compound of formula (I)has an XRD pattern substantially as shown in FIG. 1, labeled Form 1.

In certain embodiments, a crystalline compound of formula (I) is notsolvated (e.g., the crystal lattice does not comprise molecules of asolvent). In certain alternative embodiments, a crystalline compound offormula (I) is solvated.

In certain embodiments, a crystalline DMSO solvate of the compound offormula (I) has 2θ values 8.29; 17.39; 19.54; 22.78; and 22.98. Infurther embodiments, the crystalline DMSO solvate has 2θ values 8.29;17.39; 19.54; 19.74; 20.98; 21.58; 22.03; 22.78; 22.98; and 25.53. Inyet further embodiments, the crystalline DMSO solvate has 2θ values8.29; 16.10; 17.39; 19.24; 19.54; 19.74; 20.33; 20.78; 20.98; 21.18;21.58; 22.03; 22.78; 22.98; 25.53; 28.48; and 29.48. In furtherembodiments, the crystalline DMSO solvate has 2θ values 8.29; 13.12;15.79; 16.10; 16.69; 17.39; 19.03; 19.24; 19.54; 19.74; 20.33; 20.78;20.98; 21.18; 21.58; 22.03; 22.78; 22.98; 23.95; 24.14; 24.48; 24.64;25.14; 25.53; 25.87; 26.89; 27.18; 27.67; 28.02; 28.13; 28.48; 28.98;29.34; 29.48; and 29.92.

The certain embodiments, a crystalline DMSO solvate of the compound offormula (I) has an XRD pattern substantially as shown in FIG. 1, labeledForm 2.

In certain embodiments, the crystalline DMSO solvate of the compound offormula (I) contains about 1.0, about 1.1, or about 1.2 molecules ofDMSO to one molecule of NMN.

In certain embodiments, the invention relates to a pharmaceuticalcomposition comprising a crystalline compound of formula (I) and one ormore pharmaceutically acceptable excipients. In certain embodiments, thepharmaceutical composition is selected from tablets, capsules, andsuspensions.

The term “substantially pure” as used herein, refers to a crystallinepolymorph that is greater than 90% pure, meaning that contains less than10% of any other compound, including the corresponding amorphouscompound or an alternative polymorph of the crystalline salt.Preferably, the crystalline polymorph is greater than 95% pure, or evengreater than 98% pure.

Methods of Making the Crystalline Forms of NMN

In certain embodiments, the invention relates to a method for thepreparation of a crystalline compound having the structure of formula(I), comprising a) providing a mixture of a compound of formula (I) in asolvent; and b) crystallizing the compound of formula (I) from themixture comprising the compound of formula (I).

In certain embodiments, the mixture comprising the compound of formula(I) is a solution. In certain embodiments, the mixture is a slurry or asuspension.

In certain embodiments, the crystalline compound made by the methods ofthe invention is anhydrous.

In certain embodiments, the crystalline compound made by the methods ofthe invention is a solvate, e.g., a DMSO solvate.

In certain embodiments, the mixture comprising the compound of formula(I) is a solution, and the step of crystallizing the compound from themixture comprises bringing the solution to supersaturation to cause thecompound of formula (I) to precipitate out of solution.

In certain embodiments, bringing the mixture comprising the compound offormula (I) to supersaturation comprises the slow addition of ananti-solvent, such as heptanes, hexanes, ethanol, or another polar ornon-polar liquid miscible with the organic solvent, allowing thesolution to cool (with or without seeding the solution), reducing thevolume of the solution, or any combination thereof. In certainembodiments, bringing the mixture comprising the compound of formula (I)to supersaturation comprises adding an anti-solvent, cooling thesolution to ambient temperature or lower, and reducing the volume of thesolution, e.g., by evaporating solvent from the solution. In certainembodiments, allowing the solution to cool may be passive (e.g.,allowing the solution to stand at ambient temperature) or active (e.g.,cooling the solution in an ice bath or freezer).

In certain embodiments, the preparation method further comprisesisolating the crystals, e.g., by filtering the crystals, by decantingfluid from the crystals, or by any other suitable separation technique.In further embodiments, the preparation method further comprises washingthe crystals.

In certain embodiments, the preparation method further comprisesinducing crystallization. The method can also comprise drying thecrystals, for example under reduced pressure. In certain embodiments,inducing precipitation or crystallization comprises secondarynucleation, wherein nucleation occurs in the presence of seed crystalsor interactions with the environment (crystallizer walls, stirringimpellers, sonication, etc.).

In certain embodiments, the solvent comprises acetonitrile,N,N-dimethylacetamide (DMA), dimethylformamide (DMF), dimethylsulfoxide(DMSO), ethanol, ethyl acetate, heptanes, hexanes, isopropyl acetate,methanol, methylethyl ketone, N-methyl-2-pyrrolidone (NMP),tetrahydrofuran, toluene, 2-propanol, 1-butanol, water, or anycombination thereof. In certain preferred embodiments, for example toachieve Form 1, the solvent is methanol or water. In other preferredembodiments, for example to achieve Form 2, the solvent isdimethylsulfoxide.

In certain embodiments, washing the crystals comprises washing with aliquid selected from anti-solvent, acetonitrile, ethanol, heptanes,hexanes, methanol, tetrahydrofuran, toluene, water, or a combinationthereof. As used herein, “anti-solvent” means a solvent in which thesalt crystals are insoluble, minimally soluble, or partially soluble. Inpractice, the addition of an anti-solvent to a solution in which thesalt crystals are dissolved reduces the solubility of the salt crystalsin solution, thereby stimulating precipitation of the salt. In certainembodiments, the crystals are washed with a combination of anti-solventand the organic solvent. In certain embodiments, the anti-solvent iswater, while in other embodiments it is an alkane solvent, such ashexane or pentane, or an aromatic hydrocarbon solvent, such as benzene,toluene, or xylene. In certain embodiments, the anti-solvent ismethanol.

In certain embodiments, washing the crystals comprises washing thecrystalline compound of formula (I) with a solvent or a mixture of oneor more solvents, which are described above. In certain embodiments, thesolvent or mixture of solvents is cooled prior to washing.

In certain embodiments, the methods of making the crystalline forms ofNMN are used to remove one or more impurities from NMN. In certainembodiments, the crystallization methods described herein are used forpurifying NMN, e.g., as a final purification step in the manufacture ofthe compound.

Uses of Crystal Forms of NMN

NMN is produced from nicotinamide in the NAD biosynthesis pathway, areaction that is catalyzed by Nampt. NMN is further converted to NAD inthe NAD biosynthesis pathway, a reaction that is catalyzed by Nmnat.“Nicotinamide Adenine Dinucleotide” (NAD), which corresponds to thefollowing structure,

is produced from the conversion of nicotinamide to NMN, which iscatalyzed by Nampt, and the subsequent conversion of NMN to NAD, whichis catalyzed by Nmnat. In mammals, the functional homolog of yeast PNC1is NAMPT, which also catalyzes the first step in NAD salvage. NAMPTcatalyzes the formation of nicotinamide mononucleotide (NMN) from NAM,which is then converted to NAD by NMNAT1, NMNAT2, and NMNAT3.Nicotinamide riboside, a precursor to NAD, enters the salvage pathwayafter being converted to NMN by nicotinamide riboside kinase (NRK)enzymes.

Thus, diseases, disorders and conditions that are affected by increasingNAD levels are likewise affected by the amount of NMN precursoravailable for NAD biosynthesis, and thus can be treated by administeringthe NMN compounds and compositions disclosed herein.

In certain embodiments, NMN works through the nicotinamidemononucleotide adenylyltransferase (Nmnat1) pathway or other pathways ofNAD+ biosynthesis which have nutritional and/or therapeutic value inimproving plasma lipid profiles, prevention of stroke, and/or prolonginglife and well-being. Other embodiments relate to a method for preventingor treating a disease or condition associated with the nicotinamidemononucleotide adenylyltransferase (Nmnat1) pathway or other pathways ofNAD+ biosynthesis by administering a composition comprising NMN.Diseases or conditions which typically have altered levels of NAD+ orits precursors which can be prevented or treated by supplementing a dietor therapeutic treatment regime with NMN and/or NAD+ include, but arenot limited to, lipid disorders, (e.g., dyslipidemia,hypercholesterolaemia or hyperlipidemia), stroke, type I and IIdiabetes, cardiovascular disease, and other physical problems associatedwith obesity.

Neurodegenerative Diseases

Axon degeneration occurs frequently in neurodegenerative diseases andperipheral neuropathies. The degeneration of transected axons is delayedin Wallerian degeneration slow (Wlds) mice with the overexpression of afusion protein with the nicotinamide adenine dinucleotide (NAD+)synthetic enzyme, nicotinamide mononucleotide adenylyltransferase(Nmnat1). Both Wld(s) and Nmnat1 themselves are functional in preventingaxon degeneration in neuronal cultures.

NAD+ levels decrease in injured, diseased, or degenerating neural cellsand preventing this NAD+ decline efficiently protects neural cells fromcell death. See, Araki & Milbrandt “Increased nuclear NAD+ biosynthesisand SIRT1 activation prevent axonal degeneration” Science. 2004 Aug. 13;305(5686):1010-3 and Wang et al., “A local mechanism mediatesNAD-dependent protection of axon degeneration” J Cell Biol.170(3):349-55 (2005) hereby incorporated by reference in their entirety.As NMN is capable of increasing intracellular levels of NAD+, NMN isuseful as a therapeutic or nutritional supplement in managing injuries,diseases, and disorders affecting the central nervous system and theperipheral nervous system, including, but not limited to, trauma orinjury to neural cells, diseases or conditions that harm neural cells,and neurodegenerative diseases or syndromes. The correlation ofincreased NAD+ synthesis with beneficial outcomes in neural injuries anddiseases or conditions has been discussed in, e.g., Stein et al.,“Expression of Nampt in Hippocampal and Cortical Excitatory Neurons IsCritical for Cognitive Function” The Journal of Neuroscience 201434(17):5800-5815; and Stein et al., “Specific ablation of Nampt in adultneural stem cells recapitulates their functional defects during aging”EMBO J. 2014 33:1321-1340.

Some neurodegenerative diseases, neurodegenerative syndromes, diseasesand conditions that harm neural cells, or otherwise cause injury toneural cells are described below.

Essential tremor (ET) is the most common movement disorder. It is asyndrome characterized by a slowly progressive postural and/or kinetictremor, usually affecting both upper extremities.

Parkinson's disease (PD) is a progressive neurodegenerative disorderassociated with a loss of dopaminergic nigrostriatal neurons.

Alzheimer's disease (AD) is the most common form of dementia. It is aprogressive degenerative disease of the brain, strongly associated withadvanced age. Over time, people with the disease lose their ability tothink and reason clearly, judge situations, solve problems, concentrate,remember useful information, take care of themselves, and even speak. Anumber of neurodegenerative diseases such as Alzheimer's disease executetheir biological impact in the brain. It is preferred that nicotinamidemononucleotide based compounds disclosed herein are capable of passingthe blood-brain-barrier (BBB).

Huntington's disease (HD) is an incurable, adult-onset, autosomaldominant inherited disorder associated with cell loss within a specificsubset of neurons in the basal ganglia and cortex.

Ataxia is defined as an inability to maintain normal posture andsmoothness of movement. Neurologic symptoms and signs such as seizuresand movement disorders (e.g., dystonia, chorea) may accompany ataxia.

Catatonia is a state of apparent unresponsiveness to external stimuli ina person who is apparently awake.

Epilepsy is defined as a chronic condition characterized by spontaneous,recurrent seizures; seizure is defined as a clinical event associatedwith a transient, hypersynchronous neuronal discharge.

Neuroleptic malignant syndrome (NMS) refers to the combination ofhyperthermia, rigidity, and autonomic dysregulation that can occur as aserious complication of the use of antipsychotic drugs.

Chorea is an involuntary abnormal movement, characterized by abrupt,brief, nonrhythmic, nonrepetitive movement of any limb, often associatedwith nonpatterned facial grimaces. Chorea gravidarum (CG) is the termgiven to chorea occurring during pregnancy.

Cortical basal ganglionic degeneration (CBGD) clinical characteristicsinclude progressive dementia, parkinsonism, and limb apraxia.Dysfunction of the central or peripheral nervous system pathways maycause autonomic dysfunction.

Dystonia is a syndrome of sustained muscle contractions, usuallyproducing twisting and repetitive movements or abnormal postures.Writer's cramp is a form of task-specific focal dystonia.

Mental retardation (MR) is a condition in which intellectual capacity islimited significantly. Developmental disability describes a conditionthat limits an individual's ability to perform activities and roles asexpected in a certain social environment. Frequently, MR anddevelopmental disabilities are present simultaneously as a consequenceof brain damage.

Neuroacanthocytosis is a progressive neurologic disease characterized bymovement disorders, personality changes, cognitive deterioration, axonalneuropathy, and seizures. Most patients have acanthocytosis onperipheral blood smear at some point during the course of the disease.

Pelizaeus-Merzbacher disease (PMD) and X-linked spastic paraplegia type2 (SPG2) are at opposite ends of a clinical spectrum of X-linkeddiseases caused by mutations of the same gene, the proteolipid protein 1(PLP1) gene, and resulting in defective central nervous system (CNS)myelination. Clinical signs usually include some combination ofnystagmus, stridor, spastic quadriparesis, hypotonia, cognitiveimpairment, ataxia, tremor, and diffuse leukoencephalopathy on MRIscans.

Progressive supranuclear palsy (PSP), also known asSteele-Richardson-Olszewski syndrome, is a neurodegenerative diseasethat affects cognition, eye movements, and posture.

Striatonigral degeneration (SND) is a neurodegenerative disease thatrepresents a manifestation of multiple system atrophy (MSA). The othermanifestations are Shy-Drager syndrome (e.g., autonomic failurepredominates) and sporadic olivopontocerebellar degeneration (sOPCA,cerebellum predominates).

Ischemic stroke occurs due to a loss of blood supply to part of thebrain, initiating the ischemic cascade. Brain tissue ceases to functionif deprived of oxygen for more than 60 to 90 seconds and after a fewhours will suffer irreversible injury possibly leading to death of thetissue, i.e., infarction. Atherosclerosis may disrupt the blood supplyby narrowing the lumen of blood vessels leading to a reduction of bloodflow, by causing the formation of blood clots within the vessel, or byreleasing showers of small emboli through the disintegration ofatherosclerotic plaques. Embolic infarction occurs when emboli formedelsewhere in the circulatory system, typically in the heart as aconsequence of atria fibriliation, or in the carotid arteries. Thesebreak off, enter the cerebral circulation, then lodge in and occludebrain blood vessels.

Due to collateral circulation within the region of brain tissue affectedby ischemia, there is a spectrum of severity. Thus, part of the tissuemay immediately die while other parts may only be injured and couldpotentially recover. The ischemia area where tissue might recover isreferred to as the ischemic penumbra.

As oxygen or glucose becomes depleted in ischemic brain tissue, theproduction of high energy phosphate compounds such as adeninetriphosphate (ATP) fails, leading to failure of energy dependentprocesses necessary for tissue cell survival. This sets off a series ofinterrelated events that result in cellular injury and death. Theseinclude the failure of mitochondria, which can lead further towardenergy depletion and may trigger cell death due to apoptosis. Otherprocesses include the loss of membrane ion pump function leading toelectrolyte imbalances in brain cells. There is also the release ofexcitatory neurotransmitters, which have toxic effects in excessiveconcentrations.

Spinal cord injury, or myelopathy, is a disturbance of the spinal cordthat results in loss of sensation and mobility. The two common types ofspinal cord injury are: trauma: automobile accidents, falls, gunshots,diving accidents, etc. and disease: polio, spina bifida, tumors,Friedreich's ataxia, etc. It is important to note that the spinal corddoes not have to be completely severed for there to be a loss offunction. In fact, the spinal cord remains intact in most cases ofspinal cord injury.

Traumatic brain injury (TBI), also called intracranial injury, or simplyhead injury, occurs when a sudden trauma causes brain damage. TBI canresult from a closed head injury or a penetrating head injury and is oneof two subsets of acquired brain injury (ABI). The other subset isnon-traumatic brain injury (e.g., stroke, meningitis, anoxia). Parts ofthe brain that can be damaged include the cerebral hemispheres,cerebellum, and brain stem. Symptoms of a TBI can be mild, moderate, orsevere, depending on the extent of the damage to the brain. Outcome canbe anything from complete recovery to permanent disability or death. Acoma can also affect a child's brain. The damage from TBI can be focal,confined to one area of the brain, or diffuse, involving more than onearea of the brain. Diffuse trauma to the brain is frequently associatedwith concussion (a shaking of the brain in response to sudden motion ofthe head), diffuse axonal injury, or coma. Localized injuries may beassociated with neurobehavioral manifestations, hemiparesis or otherfocal neurologic deficits.

Another insult to the brain that can cause injury is anoxia. Anoxia is acondition in which there is an absence of oxygen supply to an organ'stissues, even if there is adequate blood flow to the tissue. Hypoxiarefers to a decrease in oxygen supply rather than a complete absence ofoxygen, and ischemia is inadequate blood supply, as is seen in cases inwhich the brain swells. In any of these cases, without adequate oxygen,a biochemical cascade called the ischemic cascade is unleashed, and thecells of the brain can die within several minutes. This type of injuryis often seen in near-drowning victims, in heart attack patients(particularly those who have suffered a cardiac arrest), or in peoplewho suffer significant blood loss from other injuries that then causes adecrease in blood flow to the brain due to circulatory (hypovolemic)shock.

Regulating Blood Glucose Concentration

Provided herein is a process for regulating the concentration of bloodglucose in a mammal. As utilized herein, regulating the concentration ofblood glucose refers to any increase, decrease, and/or maintenance in orof the concentration of blood glucose as compared to a previouslydetermined level.

NMN may be administered to a mammal in need of such treatment. Forexample, the mammal may require an increase in blood glucoseconcentration. Alternatively, the mammal may require a decrease in bloodglucose concentration. Or, the mammal may require maintenance of bloodglucose concentration above, at, or below a particular level or within aparticular range (e.g., through a series of increases and/or decreases,or through no increases or decreases). The blood glucoseconcentration-regulating NMN may also be administered to a mammal as aprophylactic measure; that is, the mammal is in need of treatment toprevent or delay the occurrence or onset of a medical condition such as,for example, type 1 or type 2 diabetes.

The ability to regulate the concentration of blood glucose in a mammalaccording to the processes described herein (e.g., by administering to amammal a blood glucose regulating amount of a compound of the presentinvention may be advantageous in the treatment and/or prevention of avariety of complications, diseases, and/or illnesses. The role ofincreased NAD+ levels on metabolic diseases and conditions has beendescribed in, for example, Yoshino et al., “Nicotinamide mononucleotide,a key NAD+ intermediate, treats the pathophysiology of diet- andage-induced diabetes” Cell Metab. 2011 14:528-536; and Garten, et al.,“Nampt: Linking NAD biology, metabolism, and cancer” Trends EndocrinolMetab. 2009 20(3):130-138. In general, the present invention may beutilized to treat a variety of acute, intermediate stage, and chronicconditions that may be affected by systemic NAD biosynthesis eitherdirectly or indirectly.

For example, the regulation of blood glucose concentration may beeffective in the treatment and/or prophylaxis of such medical conditionsas brain ischemia-induced hypoglycemia, hypoglycemic brain injury causedby, e.g., congenital hyperinsulinism in children, and/or otherconditions that severely reduce blood glucose levels. Alternatively, theregulation of blood glucose concentration may be effective incounteracting the effects of the injection of an excessive amount ofinsulin, or an insufficient dietary or vitamin intake (e.g.,deficiencies in vitamin B3 (niacin, which is derived from nicotinic acidand nicotinamide) can result in pellagra, the classic niacin deficiencydisease, characterized by bilateral dermatitis, diarrhea, and dementia).

Further, regulation of blood glucose concentration may be effective inthe treatment and/or prophylaxis of hypoglycemia, hyperglycemia,impaired glucose tolerance, impaired fasting glucose, and type 1 andtype 2 diabetes.

The regulation of blood glucose concentration according to the methodsdescribed herein may also be advantageous in counteracting the effectsof blood glucose concentration-decreasing drugs such as acetaminophen,alcohol, anabolic steroids, clofibrate, disopyramide, gemfibrozil,monoamine oxidase inhibitors (MAOIs), pentamidine, or sulfonylureamedications (such as glipizide, glyburide, and glimepiride).

Other conditions having a plausible connection to NAD biosynthesis, suchas dementia, may also be beneficially treated and/or prevented by bloodglucose regulation. See, e.g., Guest, et al., “Changes in OxidativeDamage, Inflammation and [NAD(H)] with Age in Cerebrospinal Fluid” PLOSOne. January 2014 9(1): e85335.

The increase, decrease, and/or maintenance of blood glucoseconcentration can be quantified, for example, by percentage above,below, or in between one or more previously determined levels, or can bequantified by a particular blood glucose concentration or a rangethereof.

For example, the blood glucose concentration may be increased to atleast about 5% above a previously determined level; to at least about10% above a previously determined level; to at least about 25% above apreviously determined level; to at least about 50% above a previouslydetermined level; to at least about 75% above a previously determinedlevel; to at least about 100% above a previously determined level; to atleast about 150% above a previously determined level; or to at leastabout 200% above a previously determined level. By way of anotherexample, the blood glucose concentration may be decreased to at leastabout 5% below a previously determined level; to at least about 10%below a previously determined level; to at least about 25% below apreviously determined level; to at least about 50% below a previouslydetermined level; to at least about 75% below a previously determinedlevel; to at least about 100% below a previously determined level; to atleast about 150% below a previously determined level; or to at leastabout 200% below a previously determined level. By way of yet anotherexample, the blood glucose concentration may be maintained (e.g., by aseries of increases and/or decreases, or by no increases and/ordecreases) at a concentration that is no more than about 50% greater orabout 50% less than a previously determined level; e.g., no more thanabout 40% greater or about 40% less; no more than about 30% greater orabout 30% less; no more than about 20% greater or about 20% less; nomore than about 10% greater or about 10% less; or no more than about 5%greater or about 5% less.

Alternatively, the blood glucose concentration may be maintained (e.g.,by a series of increases and/or decreases, or by no increases and/ordecreases) at, above, or below a particular blood glucose concentrationor within a desired range of blood glucose concentrations. For example,the blood glucose concentration may be maintained at a concentration ofgreater than about 60 mg/dL; greater than about 70 mg/dL; greater thanabout 100 mg/dL; greater than about 110 mg/dL; or greater than about 125mg/dL. Alternatively, the blood glucose concentration may be maintainedat a concentration of less than about 200 mg/dL; less than about 175mg/dL; less than about 150 mg/dL; less than about 125 mg/dL; less thanabout 110 mg/dL; or less than about 100 mg/dL. By way of anotherexample, the blood glucose concentration may be maintained at aconcentration of from about 60 mg/dL to about 140 mg/dL; from about 90mg/dL to about 130 mg/dL; from about 100 mg/dL to about 125 mg/dL; orfrom about 110 mg/dL to about 125 mg/dL.

Drug Toxicity

In some embodiments, the invention relates to the use of NMN to preventadverse effects and protect cells from toxicity. Toxicity may be anadverse effect of radiation or external chemicals on the cells of thebody. Examples of toxins are pharmaceuticals, drugs of abuse, andradiation, such as UV or X-ray light. Both radiative and chemical toxinshave the potential to damage biological molecules such as DNA. Thisdamage typically occurs by chemical reaction of the exogenous agent orits metabolites with biological molecules, or indirectly throughstimulated production of reactive oxygen species (e.g., superoxide,peroxides, hydroxyl radicals). Repair systems in the cell excise andrepair damage caused by toxins.

Enzymes that use NAD+ play a part in the DNA repair process. Forexample, DNA repair syndromes include, but are not limited to, Cockaynesyndrome. Specifically, the poly(ADP-ribose) polymerases (PARPs),particularly PARP-1, are activated by DNA strand breaks and affect DNArepair. The PARPs consume NAD+ as an adenosine diphosphate ribose (ADPR)donor and synthesize poly(ADP-ribose) onto nuclear proteins such ashistones and PARP itself. Although PARP activities facilitate DNArepair, overactivation of PARP can cause significant depletion ofcellular NAD+, leading to cellular necrosis. The apparent sensitivity ofNAD+ metabolism to genotoxicity has led to pharmacologicalinvestigations into the inhibition of PARP as a means to improve cellsurvival. Numerous reports have shown that PARP inhibition increasesNAD+ concentrations in cells subject to genotoxicity, with a resultingdecrease in cellular necrosis. See, e.g., Fang, et al., DefectiveMitophagy in XPA via PARP-1 Hyperactivation and NAD+/SIRT1 Reduction.Cell 2014 157:882-896. Nevertheless, cell death from toxicity stilloccurs, presumably because cells are able to complete apoptotic pathwaysthat are activated by genotoxicity. Thus, significant cell death isstill a consequence of DNA/macromolecule damage, even with inhibition ofPARP. This consequence suggests that improvement of NAD+ metabolism ingenotoxicity can be partially effective in improving cell survival butthat other proteins that modulate apoptotic sensitivity, such assirtuins, may also play important roles in cell responses to genotoxins.

Physiological and biochemical mechanisms that determine the effects ofchemical and radiation toxicity in tissues are complex, and evidenceindicates that NAD+ metabolism is an important aspect of cell stressresponse pathways. For example, upregulation of NAD+ metabolism, vianicotinamide/nicotinic acid mononucleotide (NMNAT) overexpression, hasbeen shown to protect against neuron axonal degeneration, andnicotinamide used pharmacologically has been recently shown to provideneuron protection in a model of fetal alcohol syndrome and fetalischemia. Such protective effects could be attributable to upregulatedNAD+ biosynthesis, which increases the available NAD+ pool subject todepletion during genotoxic stress. This depletion of NAD+ is mediated byPARP enzymes, which are activated by DNA damage and can deplete cellularNAD+, leading to necrotic death. Another mechanism of enhanced cellprotection that could act in concert with upregulated NAD+ biosynthesisis the activation of cell protection transcriptional programs regulatedby sirtuin enzymes.

Aging/Stress

In certain embodiments, the invention provides a method extending thelifespan of a cell, extending the proliferative capacity of a cell,slowing aging of a cell, promoting the survival of a cell, delayingcellular senescence in a cell, mimicking the effects of calorierestriction, increasing the resistance of a cell to stress, orpreventing apoptosis of a cell, by contacting the cell with NMN and/orNAD+. Recent studies have demonstrated the role NAD+ plays in the agingprocess and in age-related diseases and conditions. See, e.g., Imai, etal., “NAD+ and sirtuins in aging and disease” Trends in Cell Biol. 201424(8): 464-471; and Gomes, et al. “Declining NAD+ Induces aPseudohypoxic State Disrupting Nuclear-Mitochondrial Communicationduring Aging” Cell 2013 155:1624-1638.

The methods described herein may be used to increase the amount of timethat cells, particularly primary cells (e.g., cells obtained from anorganism, e.g., a human), may be kept alive in an ex vivo cell culture.Embryonic stem (ES) cells and pluripotent cells, and cellsdifferentiated therefrom, may also be treated with a nicotinamidemononucleotide based or derivative compound to keep the cells, orprogeny thereof, in culture for longer periods of time. Such cells canalso be used for transplantation into a subject, e.g., after ex vivomodification.

In certain embodiments, cells that are intended to be preserved for longperiods of time may be treated with NMN and/or NAD+. The cells may be insuspension (e.g., blood cells, serum, biological growth media, etc.) orin tissues or organs in a subject. For example, blood collected from anindividual for purposes of transfusion may be treated with NMN and/orNAD+ to preserve the blood cells for longer periods of time.Additionally, blood to be used for forensic purposes may also bepreserved using NMN and/or NAD+. Other cells that may be treated toextend their lifespan or protect against apoptosis include cells forconsumption, e.g., cells from non-human mammals (such as meat) or plantcells (such as vegetables).

NMN and/or NAD+ may also be applied during developmental and growthphases in mammals, plants, insects or microorganisms, in order to, e.g.,alter, retard or accelerate the developmental and/or growth process.

In certain embodiments, NMN and/or NAD+ may be used to treat cellsuseful for transplantation or cell therapy, including, for example,solid tissue grafts, organ transplants, cell suspensions, stem cells,bone marrow cells, etc. The cells or tissue may be an autograft, anallograft, a syngraft or a xenograft. The cells or tissue may be treatedwith NMN and/or NAD+ prior to administration/implantation, concurrentlywith administration/implantation, and/or postadministration/implantation into a subject. The cells or tissue may betreated prior to removal of the cells from the donor individual, ex vivoafter removal of the cells or tissue from the donor individual, or postimplantation into the recipient. For example, the donor or recipientindividual may be treated systemically with NMN and/or NAD+ or may havea subset of cells/tissue treated locally with NMN and/or NAD+. Incertain embodiments, the cells or tissue (or donor/recipientindividuals) may additionally be treated with another therapeutic agentuseful for prolonging graft survival, such as, for example, animmunosuppressive agent, a cytokine, an angiogenic factor, etc.

In certain embodiments, cells may be treated with an amount of NMN thatincreases the level of NAD+ in vivo, e.g., to increase their lifespan orprevent apoptosis. For example, skin can be protected from aging (e.g.,developing wrinkles, loss of elasticity, etc.) by treating skin orepithelial cells with an amount of NMN that increases the level ofintracellular NAD+. In exemplary embodiments, skin is contacted with apharmaceutical or cosmetic composition comprising an amount of NMN thatincreases the level of intracellular NAD+. Exemplary skin afflictions orskin conditions that may be treated in accordance with the methodsdescribed herein include disorders or diseases associated with or causedby inflammation, sun damage or natural aging. For example, thecompositions find utility in the prevention or treatment of contactdermatitis (including irritant contact dermatitis and allergic contactdermatitis), atopic dermatitis (also known as allergic eczema), actinickeratosis, keratinization disorders (including eczema), epidermolysisbullosa diseases (including penfigus), exfoliative dermatitis,seborrheic dermatitis, erythemas (including erythema multiforme anderythema nodosum), damage caused by the sun or other light sources,discoid lupus erythematosus, dermatomyositis, psoriasis, skin cancer andthe effects of natural aging. In other embodiments, an amount of NMNthat increases the level of intracellular NAD+ may be used for thetreatment of wounds and/or burns to promote healing, including, forexample, first-, second- or third-degree burns and/or thermal, chemicalor electrical burns. The formulations may be administered topically, tothe skin or mucosal tissue, as an ointment, lotion, cream,microemulsion, gel, solution or the like, as further described herein,within the context of a dosing regimen effective to bring about thedesired result.

Topical formulations comprising an amount of NMN that increases thelevel of intracellular NAD+ may also be used as preventive, e.g.,chemopreventive, compositions. When used in a chemopreventive method,susceptible skin is treated prior to any visible condition in aparticular individual.

In certain embodiments, an amount of NMN that increases the level ofintracellular NAD+ may be used for treating or preventing a disease orcondition induced or exacerbated by cellular senescence in a subject;methods for decreasing the rate of senescence of a subject, e.g., afteronset of senescence; methods for extending the lifespan of a subject;methods for treating or preventing a disease or condition relating tolifespan; methods for treating or preventing a disease or conditionrelating to the proliferative capacity of cells; and methods fortreating or preventing a disease or condition resulting from cell damageor death. In certain embodiments, the method does not act by decreasingthe rate of occurrence of diseases that shorten the lifespan of asubject. In certain embodiments, a method does not act by reducing thelethality caused by a disease, such as cancer.

In certain embodiments, an amount of NMN that increases the level ofintracellular NAD+ may be administered to a subject in order togenerally increase the lifespan of its cells and to protect its cellsagainst stress and/or against apoptosis. Treating a subject with NMN maybe similar to subjecting the subject to hormesis, i.e., mild stress thatis beneficial to organisms and may extend their lifespan.

An amount of NMN that increases the level of intracellular NAD+ can alsobe administered to subjects for treatment of diseases, e.g., chronicdiseases, associated with cell death, in order to protect the cells fromcell death. Exemplary diseases include those associated with neural celldeath, neuronal dysfunction, or muscular cell death or dysfunction, suchas Parkinson's disease, Alzheimer's disease, multiple sclerosis,amyotropic lateral sclerosis, and muscular dystrophy; AIDS; fulminanthepatitis; diseases linked to degeneration of the brain, such asCreutzfeld-Jakob disease, retinitis pigmentosa and cerebellardegeneration; myelodysplasis such as aplastic anemia; ischemic diseasessuch as myocardial infarction and stroke; hepatic diseases such asalcoholic hepatitis, hepatitis B and hepatitis C; joint-diseases such asosteoarthritis; atherosclerosis; alopecia; damage to the skin due to UVlight; lichen planus; atrophy of the skin; cataract; and graftrejections. Cell death can also be caused by surgery, drug therapy,chemical exposure or radiation exposure.

An amount of NMN that increases the level of intracellular NAD+ can alsobe administered to a subject suffering from an acute disease, e.g.,damage to an organ or tissue, e.g., a subject suffering from stroke ormyocardial infarction or a subject suffering from a spinal cord injury.An amount of NMN that increases the level of intracellular NAD+ may alsobe used to repair an alcoholic's liver.

Cardiovascular Disease

In certain embodiments, the invention provides methods for treatingand/or preventing a cardiovascular disease by administering to a subjectin need thereof an amount of NMN that increases the level ofintracellular NAD+. The benefits of NAD+ in treating cardiovasculardiseases has been described in several studies, such as Borradaile, etal., “NAD+, Sirtuins, and Cardiovascular Disease” Current PharmaceuticalDesign 2016 15(1):110-117.

Cardiovascular diseases that can be treated or prevented by an amount ofNMN that increases the level of intracellular NAD+ includecardiomyopathy or myocarditis; such as idiopathic cardiomyopathy,metabolic cardiomyopathy, alcoholic cardiomyopathy, drug-inducedcardiomyopathy, ischemic cardiomyopathy, and hypertensivecardiomyopathy. Also treatable or preventable using compounds andmethods described herein are atheromatous disorders of the major bloodvessels (macrovascular disease) such as the aorta, the coronaryarteries, the carotid arteries, the cerebrovascular arteries, the renalarteries, the iliac arteries, the femoral arteries, and the poplitealarteries. Other vascular diseases that can be treated or preventedinclude those related to platelet aggregation, the retinal arterioles,the glomerular arterioles, the vasa nervorum, cardiac arterioles, andassociated capillary beds of the eye, the kidney, the heart, and thecentral and peripheral nervous systems.

Yet other disorders that may be treated with an amount of NMN thatincreases the level of intracellular NAD+ include restenosis, e.g.,following coronary intervention, and disorders relating to an abnormallevel of high density and low density cholesterol. Another disease thatcan benefit from NAD+ treatment is nonalcoholic steatohepatitis (NASH)which a fatty liver disease.

Circadian Rhythm

The circadian clock is encoded by a transcription-translation feedbackloop that synchronizes behavior and metabolism with the light-darkcycle. It has been unexpectedly discovered that both the rate-limitingenzyme in mammalian NAD+ biosynthesis, nicotinamidephosphoribosyltransferase (NAMPT), and levels of NAD+, display circadianoscillations which are regulated by the core clock machinery in mice.Inhibition of NAMPT promotes oscillation of the clock gene Per2 byreleasing CLOCK:BMAL1 from suppression by SIRT1. In turn, the circadiantranscription factor CLOCK binds to and up-regulates Nampt, thuscompleting a feedback loop involving NAMPT/NAD+ and SIRT1/CLOCK:BMAL1.See, e.g., Ramsey et al., “Circadian clock feedback cycle throughNAMPT-mediated NAD+ biosynthesis” Science 2009 324:651-654.

Thus, the periodic variation in NAMPT-mediated NAD+ biosynthesissuggests that it impacts physiologic cycles and possibly the sleep-wakeand fasting-feeding cycle. Without being bound by a single theory, it isbelieved that NAD+ serves as a critical “metabolic oscillator” for therhythmic regulation of response to environmental cues through control ofSIRT1 activity. Compounds disclosed herein may be used to affect acircadian feedback loop through NAMPT-mediated NAD+ biosynthesis and/ora pathway underlying the temporal coupling of metabolic, physiologic,and circadian cycles in mammals.

The recognition of a regulatory pathway involvingNAMPT/NAD+-SIRT1/CLOCK:BMAL1 has broad implications for understandinghow physiologic and behavioral cycles are coordinated with theenvironmental light-dark cycle. For instance, during sleep, when animalsare normally quiescent and fasting, the levels of NAMPT steadilyincrease, peaking at the beginning of the wakefulness period andcoinciding with feeding. As a result of the increase in NAMPT, NAD+rises to stimulate SIRT1, which orchestrates an appropriate metabolicresponse in liver involving a switch from catabolic to anabolicpathways.

In certain embodiments, the present invention provides methods forregulation of the core clock machinery (sometimes also referred to asthe circadian clock) of a mammal, thereby affecting behaviors,activities, and/or biological functions that occur in or are affected bya diurnal or circadian cycle and that are regulated, at least in part,by the circadian clock. Generally, the methods involve theadministration of a therapeutic or prophylactic amount of a circadianclock-regulating compound to a patient or mammal in need of regulationof the circadian clock.

The methods of treatment disclosed herein are generally directed tomethods of regulating the circadian clock, thereby regulating oraffecting biological functions that are regulated by (sometimes alsosaid to be affected by, affiliated with, or mediated by) the activity ofthe circadian clock. Typically, these biological functions display apattern of activity and inactivity that is generally repeatedapproximately every 24 hours, oscillating between “active” and“inactive” states during the 24 hour period.

Thus, the present invention provides methods of regulating the activityof the circadian clock by administering to a mammal in need thereof acircadian-clock regulating compound. Generally, the regulation of theactivity of the circadian clock is the result of the regulation ofCLOCK:BMAL1, which is achieved according to the present methods byregulating the activity of SIRT1. The activity of SIRT1 is generallyregulated according to the present methods by administration of acircadian clock-regulating compound, and in certain embodiments, byadministration of an amount of NMN that affects the NAD+ pathway. Theregulation of the circadian clock thereby permits regulation ofactivities mediated by the circadian clock.

According to the present invention, the activity of the circadian clockmay be increased, decreased, or maintained by the administration of acircadian clock-regulating compound. Accordingly, biological functions(sometimes also referred to as biological activities) that are regulatedby the activity of the circadian clock may also be increased, decreased,or maintained. In addition, these biological functions may also be timeshifted; that is to say, an activity that typically occurs during aparticular period, such as for example, during daytime or daylight hours(sometimes also referred to as the light cycle) or during the night ornighttime hours (sometimes also referred to as the dark cycle) may beshifted such that the activity occurs during the dark or light cycle,respectively, instead.

Any of a number of biological functions that are typically affected bythe activity of the circadian clock may be regulated by the methods ofthe present invention. Thus, the present methods may be used to treatdisorders or disease states that are the result of, for example, theirregular, inadequate, or pathological function of the circadian clock.Similarly, the present methods may be used to treat disorders orsymptomatology caused by exogenous factors that affect the properfunction or activity of the circadian clock or that require a“resetting” of the clock. For example, administration of circadianclock-regulating compound to a patient experiencing a metabolic disorderprovides therapeutic benefit not only when the patient's serum NMN orNAD level is increased, but also when an improvement is observed in thepatient with respect to other disorders that accompany the metabolicdisorder, like weight loss or gain. In some treatment regimens, thecircadian clock-regulating compound of the invention may be administeredto a patient at risk of developing a disorder as described herein or toa patient reporting one or more of the physiological symptoms of such adisorder, even though a diagnosis of a metabolic disorder may not havebeen made.

Examples of disorders, disease states, or symptomatology that may betreated according to the methods of the present invention include, butare not limited to, travel to or across one or more time zones, a changein work shifts, night shift work, or a change in the physical status ofa mammal caused by, for example, pregnancy or administration ofmedications of any kind. Accordingly, the methods of the presentinvention may be used to treat or prevent disorders, symptoms ofdisorders, or symptoms caused by exogenous factors. Such disorders andsymptoms may include, for example, metabolic disorders, such as impropercycling or timing of feeding and fasting cycles, hyperglycemia,hypoglycemia, or diabetes; sleep disorders, such as insomnia, advancedsleep phase syndrome, delayed sleep phase syndrome, inconsistentsleep/wake cycles, or narcolepsy or to improve wakefulness inindividuals suffering from excessive sleepiness; and symptoms caused byexogenous factors, such as, travel to or across one or more time zones(jet lag), shifting into or out of daylight savings time, a change inwork shifts or night shift work, pregnancy, or medications being takenfor unrelated diseases or disorders.

Accordingly, in certain embodiments, the present invention is directedto a method of regulating a biological function in a mammal, thefunction being affected by the circadian clock. The method comprisesadministering a therapeutic or prophylactic (sometimes also referred toas a circadian clock-regulating) amount of a circadian clock-regulatingcompound to the mammal. The biological function can be, for example, anyone of the biological functions described herein. In certainembodiments, the invention comprises a method of treating a metabolicdisorder in a mammal and comprises administering a therapeutic orprophylactic amount of a circadian clock-regulating compound to themammal. In other embodiments, the invention comprises a method oftreating a disorder in a mammal mediated by the function of thecircadian clock and comprises administering a therapeutic orprophylactic amount of a circadian clock-regulating compound to themammal. According to any one of these embodiments, the circadianclock-regulating compound may be, for example, nicotinamide,nicotinamide mononucleotide (NMN), nicotinamide adenine dinucleotide(NAD); salts and prodrugs thereof, nicotinamidephosphoribosyltransferase (NAMPT); and combinations thereof, asdescribed in greater detail below. In other embodiments, the circadianclock-regulating compound may be an antagonist of any one of thecompounds listed above, thereby exacting an effect opposite that ofnicotinamide, nicotinamide mononucleotide (NMN), nicotinamide adeninedinucleotide (NAD); salts and prodrugs thereof; nicotinamidephosphoribosyltransferase (NAMPT); and combinations thereof.

In certain embodiments, the present invention is directed to a method ofregulating metabolic activity of a mammal comprising administering tothe mammal a therapeutic amount of a circadian clock-regulatingcompound. In certain embodiments, the metabolic activity of the mammalis increased. In other embodiments, the metabolic activity is decreased.In yet other embodiments, the metabolic activity of the mammal ismaintained at a desired level, thereby preventing fluctuations inactivity/inactivity. In still other embodiments, the metabolic activityis caused to occur in the light cycle (as opposed to its typicaloccurrence in the dark cycle). In other embodiments, the metabolicactivity is caused to occur in the dark cycle (as opposed to its typicaloccurrence in the light cycle). In certain embodiments, the circadianclock-regulating compound is administered to the mammal in order toincrease the anabolic activity of the liver (e.g., increase the activityof the metabolic pathways of the liver or shift or switch liver activityfrom catabolism to anabolism). In other embodiments, the circadianclock-regulating compound is administered to the mammal in order toincrease the catabolic activity of the liver (e.g., decrease theactivity of the metabolic process).

Mitochondrial Diseases and Metabolic Effects

In addition to regulating circadian rhythms and protect neural cellsfrom cell death, sirtuins such as SIRT3, SIRT4, and SIRT5 are found inmitochondria. SIRT3 is expressed at high levels in metabolically activetissue. Modulation of SIRT3 has a variety of physiological applicationsfor muscle cells including mimicking calorie restriction or exercise,increasing mitochodrial biogenesis or metabolism, sensitizing a cell toglucose uptake, increasing fatty acid oxidation, and decreasing reactiveoxygen species. In addition, SIRT3 is demonstrated herein to be involvedin promoting cell survival during genotoxic stress. Thus modulation ofSIRT3 levels also has applications in mediating cell survival.

Increasing the protein or activity level of SIRT3 in a muscle cell canmimic the benefits of calorie restriction or exercise. In someembodiments, the invention relates to methods for increasingmitochondrial biogenesis or metabolism or for boosting mitochondrialactivity/endurance in a muscle cell by contacting a muscle cell with anagent IS that increases the protein or activity level of SIRT3 in thecell. In some embodiments, the invention relates to methods forsensitizing a muscle cell to glucose uptake by contacting a muscle cellwith an agent that increases the protein or activity level of SIRT3 inthe cell. Further embodiments of the invention relate to methods forincreasing fatty acid oxidation in a muscle cell by contacting a musclecell with an agent that increases the protein or activity level of SIRT3in the cell. Some embodiments of the invention relate to methods fordecreasing reactive oxygen species (ROS) in a muscle cell by contactingthe muscle cell with an agent that increases the protein or activitylevel of SIRT3 in the cell.

Increasing levels of SIRT3 benefits many diseases and disorders affectedby metabolism within mitochondria. Increasing SIRT3 may be useful in anysubjects in need of metabolic activation of one or more of theirmuscles, e.g., smooth muscles or cardiac muscles or muscle cellsthereof. A subject may be a subject having cachexia or muscle wasting.

Increasing SIRT3 may also be used to increase or maintain bodytemperature, e.g., in hypothermic subjects. Alternatively, inhibitingSIRT3 may be used to reduce body temperature, e.g., in subjects havingfever or hyperthermia.

Generally, activation of SIRT3 may be used to stimulate the metabolismof any type of muscle, e.g., muscles of the gut or digestive system, orthe urinary tract, and thereby may be used to control gut motility,e.g., constipation, and incontinence.

Other embodiments in which it would be useful to increase SIRT3 includerepair of muscle, such as after a surgery or an accident, increase ofmuscle mass; and increase of athletic performance.

Thus the invention provides methods in which beneficial effects areproduced by contacting one or more muscle cells with an amount of NMNthat increases the protein or activity level of SIRT3 in the cell. Thesemethods effectively facilitate, increase or stimulate one or more of thefollowing: mimic the benefits of calorie restriction or exercise in themuscle cell, increase mitochondrial biogenesis or metabolism, increasemitochondrial activity and/or endurance in the muscle cell, sensitizethe muscle cell to glucose uptake, increase fatty acid oxidation in themuscle cell, decrease reactive oxygen species (ROS) in the muscle cell,increase PGC-1a and/or ucp3 and/or GLUT4 expression in the muscle cell,and activate AMP activated protein kinase (AMPK) in the muscle cell.

Various types of muscle cells can be contacted in accordance with theinvention. In some embodiments, the muscle cell is a skeletal musclecell. In certain embodiments, the muscle cell is a cell of a slow-twitchmuscle, such as a soleus muscle cell. The methods of the inventioninclude, in some embodiments, administering, to a subject in need ofsuch treatment, an amount of NMN that increases the protein or activitylevel of SIRT3 in cells of the subject.

The cell that is contacted or the subject that is treated in theaforementioned methods preferably is a cell in need of SIRT3 increase inprotein or activity level. In certain embodiments, the cell is adiseased cell of a subject.

Also provided are methods for regulating skeletal muscle metabolism orskeletal muscle energy homeostasis in a subject. In such methods, anagent that modulates the protein or activity level of SIRT3 in thesubject, i.e., the SIRT3 modulators described herein, is administered toa subject in need thereof.

Also provided are methods for increasing the protein level of SIRT3 in amuscle cell or in muscles of a subject. Such methods include subjectinga cell or a subject to caloric restriction or fasting, or administeringto a subject in need thereof an amount of NMN that increases the proteinor activity level of SIRT3 in a muscle cell. Diseases, disorders andconditions in which such methods are useful include mitochondrialdiseases, metabolic disorders, neurologic disorders, muscular disorders,cardiovascular diseases, and excessive weight or obesity. Specificmetabolic disorders, diseases or conditions include insulin resistance,diabetes, diabetes related conditions or disorders, or metabolicsyndrome. Other metabolic disorders will be known to the skilled person.

Mitochondrial diseases that can be treated include diseases that show avariety of symptoms caused by dysfunction of mitochondria in cells. Themitochondrial diseases may be classified in various ways by biochemicalabnormalities, clinical symptoms or types of DNA abnormalities. Typesnamed as KSS (chronic progressive external ophthalmoplegia), MERRF(myoclonus epilepsy associated with ragged-red fibers; Fukuharasyndrome), MELAS, Leber's disease, Leigh encephalopathia and Pearson'sdisease are widely known. Among them, MELAS is a type mainly showingstroke-like episodes, occupies 30% or more of the whole and is believedto be the most frequent type in the mitochondrial disease.

In certain embodiments, NMN is useful in treating diseases or disordersassociated with DNA repair defects or mitochondrial dysfunction (e.g.,resulting from the deregulation of mitochondrial homeostasis). In someembodiments, “mitochondrial dysfunction” or “deregulation ofmitochondrial homeostasis” means that one or more mitochondrialcomponent (e.g., ETC component) is depleted, for example by a decreasein mitochondrial gene expression or mitochondrial DNA content, resultingin compromised mitochondrial function (e.g., loss of or decreasedoxidative phosphorylation (OXPHOS) capacity). Examples of DNA repairdiseases include Cockayne syndrome and TTD.

Retinal Diseases and Disorders

Photoreceptor neuronal cell death and vision can be rescued by NMNadministration. In certain embodiments, nicotinamide phosphoribosyltransferase (NAMPT)-mediated NAD biosynthesis can play a role in for rodand/or cone PR neuron survival. In certain embodiments, decreased NADlevels can cause impaired mitochondrial function in PR neurons,alterations in TCA cycle metabolites, and can lead to cell death andblindness.

Deleting NAMPT can lead to photoreceptor death, loss of normal retinalstructure and function, and vision loss. In some cases, damage tophotoreceptor neurons and their function can be reversed withsupplementation of NMN, an NAMPT enzymatic reaction product. Disclosedherein are methods of administering NMN to restore NAD levels in theretina. In some embodiments, NMN supplementation can be an effectivetherapeutic intervention for many retinal degenerative diseases.

Provided herein are methods of treating, preventing, and reducing riskof diseases associated with photoreceptor dysfunction, including,without limitation, age-related macular degeneration (AMD), inheritedand acquired retinal diseases such as, without limitation, retinitispigmentosa (RP), rod and cone dystrophism, and Leber's congenitalamaurosis (LCA) by administration of NMN to a subject. In certainembodiments, NMN administration can be an effective intervention for theprevention and/or treatment of orphan retinal degenerative diseasesincluding but not limited to rod dystrophy, cone dystrophy, retinitispigmentosa, other inherited retinal degenerations, Leber's congenitalamaurosis (LCA) and acquired retinal degenerations such as, but notlimited to, age-related macular degeneration, photoreceptor degenerationfollowing retinal detachment.

In some embodiments, these methods can comprise administering to asubject a pharmaceutically effective amount of nicotinamidemononucleotide (NMN). In some embodiments, a pharmaceutically effectiveamount of nicotinamide mononucleotide (NMN) can be an amount effectivefor increasing retinal NAD levels.

Disclosed herein are methods of treating macular degeneration in asubject. In some embodiments, the methods include treating aberrantretinal NAD levels in a subject, including aberrantly low retinal NADlevels. These methods comprise administering NMN to a subject. In someembodiments, the methods include treating retinal degeneration in asubject. In some embodiments, the methods include treating photoreceptordamage in a subject. In some embodiments, the methods include treatingphotoreceptor degeneration in a subject.

In some embodiments, the methods include treating vision loss associatedwith retinal degeneration in a subject. In some embodiments, the methodsinclude treating aberrant retinal structure in a subject. In someembodiments, the methods include increasing retinal NAD levels in asubject.

In some embodiments, the methods include reducing the risk of developingmacular degeneration in a subject. In some embodiments, the methodsinclude reducing risk of developing aberrant retinal NAD levels in asubject. In some embodiments, the methods include reducing the risk ofdeveloping retinal degeneration in a subject. In some embodiments, themethods include reducing the risk of developing photoreceptordamage/degeneration in a subject. In some embodiments, the methodsinclude reducing the risk of developing vision loss associated withretinal degeneration in a subject. In some embodiments, the methodsinclude reducing the risk of developing aberrant retinal structure in asubject.

In some embodiments, the methods include treating a retina disease in asubject. In some embodiments, a retinal disease that can be treated byadministration of NMN can be retinitis pigmentosa (RP), Leber'scongenital amaurosis (LCA), rod dystrophy, cone dystrophy, rod-conedystrophy, cone-rod dystrophy, age-related macular degeneration,photoreceptor degeneration following retinal detachments, or acombination thereof.

In certain embodiments, the crystal forms of β-nicotinamidemononucleotide (NMN) (and formulations prepared using such crystalforms) may be used for treating, ameliorating, mitigating, slowing,arresting, preventing or reversing age-associated obesity in a subject.In some embodiments, the invention relates to methods of treating,ameliorating, mitigating, slowing, arresting, preventing or reversingage-associated increases in blood lipid levels in a subject. In someembodiments, the invention relates to methods of treating, ameliorating,mitigating, slowing, arresting, preventing or reversing age-associatedloss of insulin sensitivity in a subject. In some embodiments, theinvention relates to methods of treating, ameliorating, mitigating,slowing, arresting, preventing or reversing age-associated impairment ofmemory function in a subject. In some embodiments, the invention relatesto methods of treating, ameliorating, mitigating, slowing, arresting,preventing or reversing age-associated decline in eye function in asubject. In some embodiments, the invention relates to methods oftreating, ameliorating, mitigating, slowing, arresting, preventing orreversing age-associated retinal degeneration in a subject. In someembodiments, the invention relates to methods of treating, ameliorating,mitigating, slowing, arresting, preventing or reversing dry eye. In someembodiments, the invention relates to methods of treating, ameliorating,mitigating, slowing, arresting, preventing or reversing age-associateddry eye. In some embodiments, the invention relates to methods oftreating, ameliorating, mitigating, slowing, arresting, preventing orreversing infertility.

In some embodiments, the invention provides methods of treatingage-associated defects in neural stem/progenitor cell (NSPC)functionality in a subject through administration of a crystal form ofNMN or a formulation prepared using such a crystal form. In someembodiments, the invention provides methods of reducing age-associateddecrease in a NSPC population in a subject through administration of acrystal form of NMN or a formulation prepared using such a crystal form.In some embodiments, the invention provides methods of maintaining atleast one NSPC in a subject through administration of a crystal form ofNMN or a formulation prepared using such a crystal form. In someembodiments, the invention provides methods of enhancing NADbiosynthesis in a subject through administration of a crystal form ofNMN or a formulation prepared using such a crystal form. In someembodiments, the invention provides methods of promoting NSPCproliferation in a subject, in which the methods comprise administrationof a crystal form of NMN, or a formulation prepared using such a crystalform, to the subject. The methods of each of these embodiments cancomprise, consist essentially of, or consist of administration of atherapeutically effective amount of a crystal form of NMN or aformulation prepared using such a crystal form.

In some embodiments, the invention provides methods of increasing bonedensity levels in a subject. In some embodiments, the invention providesmethods of treating aberrantly low bone density levels in a subject. Insome embodiments, the invention provides methods of treating anage-associated bone density decrease in a subject. In some embodiments,the invention provides methods of treating osteoporosis in a subject. Insome embodiments, the invention provides methods of preventing anage-associated bone density decrease in a subject. The methods of eachof these embodiments can comprise, consist essentially of, or consist ofadministration of a therapeutically effective amount of a crystal formof NMN or a formulation prepared using such a crystal form.

In certain embodiments, the invention relates to methods of preventing,methods of reducing risk of, and methods of treating various diseasesassociated with photoreceptor dysfunction, including, withoutlimitation, age-related macular degeneration (AMD), inherited andacquired retinal diseases such as, without limitation, retinitispigmentosa (RP), rod and cone dystrophism, and Leber's congenitalamaurosis (LCA) by administration of NMN. In various embodiments,administration of an NMN crystal form or a formulation prepared usingsuch a crystal form can be an effective intervention for the preventionand/or treatment of orphan retinal degenerative diseases including butnot limited to rod dystrophy, cone dystrophy, retinitis pigmentosa,other inherited retinal degenerations, Leber's congenital amaurosis(LCA) and acquired retinal degenerations such as, but not limited to,age-related macular degeneration photoreceptor degeneration followingretinal detachment. In various embodiments, a crystal form of NMN or aformulation prepared using such a crystal form can be administered byany administration route known to skilled artisans, such as, withoutlimitation, oral, parenteral, intraocular, intraperitoneal, intravenousor intramuscular routes. In various embodiments, NMN can be administeredwith or without an excipient.

In some embodiments, NMN treats an age-related disease. In certainembodiments, the age-related disease is Alzheimer's disease, amniotropiclateral sclerosis, arthritis, atherosclerosis, cachexia, cancer, cardiachypertrophy, cardiac failure, cardiac hypertrophy, cardiovasculardisease, cataracts, colitis, chronic obstructive pulmonary disease,dementia, diabetes mellitus, frailty, heart disease, hepatic steatosis,high blood cholesterol, high blood pressure, Huntington's disease,hyperglycemia, hypertension, infertility, inflammatory bowel disease,insulin resistance disorder, lethargy, metabolic syndrome, musculardystrophy, multiple sclerosis, neuropathy, nephropathy, obesity,osteoporosis, Parkinson's disease, psoriasis, sarcopenia, sleepdisorders, sepsis and/or stroke. In some embodiments, the mitochondrialdisease is mitochondrial myopathy, diabetes mellitus and deafness (DAD),Leber's hereditary optic neuropathy (LHON), Leigh syndrome, neuropathy,ataxia, retinitis pigmentosa and petosis (NARP), myoclonic epilepsy withragged red fibers (MERRF), myoneurogenic gastrointestinal encephalopathy(MNGIE), mitochondrial myopathy, encephalomyopathy, lactic acidosis,stroke-like symptoms (MELAS), Kearns-Sayre syndrome (KSS), chronicprogressive external opthalmoplegia (CPEO) and/or mtDNA depletion.

Examples of diseases, disorders, or conditions associated withmitochondrial dysfunction include, but are not limited to, aging,aging-related diseases, mitochondrial diseases (e.g., Alper's disease,Barth syndrome, beta-oxidation defects, carnitine-acyl-carnitinedeficiency, carnitine deficiency, creatine deficiency syndromes,co-enzyme Q10 deficiency, complex I deficiency, complex II deficiency,complex III deficiency, complex IV deficiency/COX deficiency, complex Vdeficiency, chronic progressive external ophthalmoplegia syndrome, CPT Ideficiency, CPT II deficiency, Kearns-Sayre syndrome, lactic acidosis,long-chain acyl-CoA dehydrongenase deficiency, Leigh syndrome, Luftdisease, glutaric aciduria type II, mitochondrial cytopathy,mitochondrial DNA depletion, mitochondrial encephalopathy, mitochondrialmyopathy, and Pearson syndrome), metabolic diseases and disorders (e.g.,amino acid deficiency), diseases resulting from mitochondrial and energydeficiency, lethargy, heart disorders, cardiovascular disease, stroke,infarction, pulmonary hypertension, ischemia, cachexia, sarcopenia,neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson'sdisease, Huntington's disease), dementia, lipodystrophy, liversteatosis, hepatitis, cirrhosis, kidney failure, preeclampsia, maleinfertility, obesity, diabetes (e.g., diabetes type I), muscledisorders, and muscle wasting.

In some embodiments, NMN is useful for promoting cell viability (invarious species), vascular remodeling, wound healing and healing ingeneral (e.g., treating wounds resulting from cuts, scrapes, surgery,bodily insults, trauma, burns, abrasions, sunburns, etc.). In someembodiments, the methods and compositions are useful for promoting ironhomeostasis and/or erythropoiesis. In some embodiments, methods andcompositions provided herein are useful to promote successful organ andtissue transplantation, or to promote recovery from organ and tissuetransplantation. In some embodiments, provided methods and compositionsare useful for preserving cells and organs. In some embodiments, methodsand compositions provided herein have cosmetic applications, for examplefor treating conditions associated with mitochondrial dysfunction whichrelate to the skin or scalp/hair, such as skin aging (e.g., loss involume and elasticity, discoloration, liver spots (lentigo senislis)),wrinkles, baldness, and loss of hair pigmentation. In some embodiments,agents or compositions described herein are useful for products ormethods relating to cosmetics, energy drinks, and/or animal and plantindustries (e.g. livestock, pets and agricultural products).

Subjects that may be treated as described herein include eukaryotes,such as mammals, e.g., humans, ovines, bovines, equines, porcines,canines, felines, non-human primate, mice, and rats. Cells that may betreated include eukaryotic cells, e.g., from a subject described above,or plant cells, yeast cells and prokaryotic cells, e.g., bacterialcells. For example, NMN may be administered to farm animals to improvetheir ability to withstand farming conditions longer.

The compound may also be used to increase lifespan, stress resistance,and resistance to apoptosis in plants. In certain embodiments, NMN isapplied to plants, e.g., on a periodic basis, or to fungi. In otherembodiments, plants are genetically modified to produce NMN. In otherembodiments, plants and fruits are treated with NMN prior to picking andshipping to increase resistance to damage during shipping. Plant seedsmay also be contacted with NMN, e.g., to preverse them.

NMN may also be used to increase lifespan, stress resistance andresistance to apoptosis in insects. In certain embodiments, NMN would beapplied to useful insects, e.g., bees and other insects that areinvolved in pollination of plants. In preferred embodiments, NMN wouldbe applied to bees involved in the production of honey. Generally, themethods described herein may be applied to any organism, e.g.,eukaryote, that may have commercial importance. For example, they can beapplied to fish (aquaculture), shrimp, pigs, and birds (e.g., chickenand fowl).

Other uses of NMN include favorably modulating the microbiome or servingas a form of vitamin B3. As the precursor of NAD, NMN could also servein assays for diseases and conditions affected by NAD biosynthesis, suchas use as a standard.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence or frequency of the disorder or condition in the treatedsample relative to an untreated control sample, or delays the onset orreduces the severity of one or more symptoms of the disorder orcondition relative to the untreated control sample. Thus, prevention ofcancer includes, for example, reducing the number of detectablecancerous growths in a population of patients receiving a prophylactictreatment relative to an untreated control population, and/or delayingthe appearance of detectable cancerous growths in a treated populationversus an untreated control population, e.g., by a statistically and/orclinically significant amount. Prevention of an infection includes, forexample, reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “treating” includes prophylactic and/or therapeutic treatments.The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

Pharmaceutical Compositions

In certain embodiments, the present invention relates to pharmaceuticalcompositions comprising a crystalline compound of formula (I) and one ormore pharmaceutically acceptable excipients, as well as formulationsprepared using such a crystalline compound and one or morepharmaceutically acceptable excipients. In certain embodiments, thepharmaceutical preparations may be for use in treating or preventing acondition or disease as described herein. In certain embodiments, thepharmaceutical preparations have a low enough pyrogen activity to besuitable for intravenous use in a human patient. In certain embodiments,the invention also relates to preparations suitable for nutraceutical,veterinary, and agriculturally-relevant uses.

Exemplary pharmaceutically acceptable excipients are presented herein,and include, for example binders, disintegrating agents, lubricants,corrigents, solubilizing agents, suspension aids, emulsifying agents,coating agents, cyclodextrins, and/or buffers. Although the dosage willvary depending on the symptoms, age and body weight of the patient, thenature and severity of the disorder to be treated or prevented, theroute of administration and the form of the drug, in general, a dailydosage of from 0.01 to 3000 mg of the compound is recommended for anadult human patient, and this may be administered in a single dose or individed doses. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.

The precise time of administration and/or amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given patient will depend upon the activity, pharmacokinetics, andbioavailability of a particular compound, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage, and type of medication),route of administration, etc. However, the above guidelines can be usedas the basis for fine-tuning the treatment, e.g., determining theoptimum time and/or amount of administration, which will require no morethan routine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

In certain embodiments, the individual to which the composition isadministered is a mammal such as a human, or a non-human mammal. Whenadministered to an animal, such as a human, the composition or thecompound is preferably administered as a pharmaceutical compositioncomprising, for example, a compound of the invention and apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are well known in the art and include, for example, aqueoussolutions such as water or physiologically buffered saline or othersolvents or vehicles such as glycols, glycerol, oils such as olive oil,or injectable organic esters. In a preferred embodiment, when suchpharmaceutical compositions are for human administration, particularlyfor invasive routes of administration (i.e., routes, such as injectionor implantation, that circumvent transport or diffusion through anepithelial barrier), the aqueous solution is pyrogen-free, orsubstantially pyrogen-free. The excipients can be chosen, for example,to effect delayed release of an agent or to selectively target one ormore cells, tissues or organs. The pharmaceutical composition can be indosage unit form such as tablet, capsule (including sprinkle capsule andgelatin capsule), granule, lyophile for reconstitution, powder,solution, syrup, suppository, injection or the like. The composition canalso be present in a transdermal delivery system, e.g., a skin patch.The composition can also be present in a solution suitable for topicaladministration, such as an eye drop, through ophthalmic mucous membraneadministration.

A pharmaceutically acceptable carrier can contain physiologicallyacceptable agents that act, for example, to stabilize, increasesolubility or to increase the absorption of a compound such as acompound of the invention. Such physiologically acceptable agentsinclude, for example, carbohydrates, such as glucose, sucrose ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins or other stabilizers orexcipients. The choice of a pharmaceutically acceptable carrier,including a physiologically acceptable agent, depends, for example, onthe route of administration of the composition. The preparation orpharmaceutical composition can be a self-emulsifying drug deliverysystem or a self-microemulsifying drug delivery system. Thepharmaceutical composition (preparation) also can be a liposome or otherpolymer matrix, which can have incorporated therein, for example, acompound of the invention. Liposomes, for example, which comprisephospholipids or other lipids, are nontoxic, physiologically acceptableand metabolizable carriers that are relatively simple to make andadminister.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations. In certain embodiments, pharmaceutical compositions of thepresent invention are non-pyrogenic, i.e., do not induce significanttemperature elevations when administered to a patient.

The term “pharmaceutically acceptable salt” refers to the relativelynon-toxic, inorganic and organic acid addition salts of the compounds.These salts can be prepared in situ during the final isolation andpurification of the compounds, or by separately reacting a purifiedcompound in its free base form with a suitable organic or inorganicacid, and isolating the salt thus formed. Representative salts includethe hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, naphthylate, mesylate, glucoheptonate, lactobionate,laurylsulphonate salts, and amino acid salts, and the like. Preparationof the crystalline salts is detailed in the Examples, below (See, forexample, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19.).

In other cases, the compounds useful in the methods of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable bases. The term “pharmaceutically acceptablesalts” in these instances refers to the relatively non-toxic inorganicand organic base addition salts of a compound. These salts can likewisebe prepared in situ during the final isolation and purification of thecompound, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate, orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary, ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum salts,and the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, and the like (see, forexample, Berge et al., supra).

A pharmaceutical composition (preparation) can be administered to asubject by any of a number of routes of administration including, forexample, orally (for example, drenches as in aqueous or non-aqueoussolutions or suspensions, tablets, capsules (including sprinkle capsulesand gelatin capsules), boluses, powders, granules, pastes forapplication to the tongue); absorption through the oral mucosa (e.g.,sublingually); anally, rectally or vaginally (for example, as a pessary,cream or foam); parenterally (including intramuscularly, intravenously,subcutaneously or intrathecally as, for example, a sterile solution orsuspension); nasally; intraperitoneally; subcutaneously; transdermally(for example as a patch applied to the skin); and topically (forexample, as a cream, ointment or spray applied to the skin, or as an eyedrop). The compound may also be formulated for inhalation. In certainembodiments, a compound may be simply dissolved or suspended in sterilewater. Details of appropriate routes of administration and compositionssuitable for same can be found in, for example, U.S. Pat. Nos.6,110,973, 5,763,493, 5,731,000, 5,541,231, 5,427,798, 5,358,970 and4,172,896, as well as in patents cited therein.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association an active compound, such as a compound ofthe invention, with the carrier and, optionally, one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association a compound of the present inventionwith liquid carriers, or finely divided solid carriers, or both, andthen, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules (including sprinkle capsules and gelatin capsules),cachets, pills, tablets, lozenges (using a flavored basis, usuallysucrose and acacia or tragacanth), lyophile, powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouthwashes and the like, each containinga predetermined amount of a compound of the present invention as anactive ingredient. Compositions or compounds may also be administered asa bolus, electuary or paste.

To prepare solid dosage forms for oral administration capsules(including sprinkle capsules and gelatin capsules), tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; (10) complexing agents,such as, modified and unmodified cyclodextrins; and (11) coloringagents. In the case of capsules (including sprinkle capsules and gelatincapsules), tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions, such as dragees, capsules (including sprinkle capsules andgelatin capsules), pills and granules, may optionally be scored orprepared with coatings and shells, such as enteric coatings and othercoatings well known in the pharmaceutical-formulating art. They may alsobe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile, otherpolymer matrices, liposomes and/or microspheres. They may be sterilizedby, for example, filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions that can be dissolved in sterile water, or some othersterile injectable medium immediately before use. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions that can be used includepolymeric substances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Liquid dosage forms useful for oral administration includepharmaceutically acceptable emulsions, lyophiles for reconstitution,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, cyclodextrins and derivatives thereof, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the compositions of the present invention canalso include adjuvants such as wetting agents, lubricants, emulsifyingand suspending agents such as sodium lauryl sulfate and magnesiumstearate, or sweetening, flavoring, coloring, perfuming, preservative,or anti-oxidant agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions for rectal, vaginal, orurethral administration may be presented as a suppository, which may beprepared by mixing one or more active compounds with one or moresuitable nonirritating excipients or carriers comprising, for example,cocoa butter, polyethylene glycol, a suppository wax or a salicylate,and which is solid at room temperature, but liquid at body temperatureand, therefore, will melt in the rectum or vaginal cavity and releasethe active compound.

Formulations of the pharmaceutical compositions for administration tothe mouth may be presented as a mouthwash, or an oral spray, or an oralointment.

Alternatively or additionally, compositions can be formulated fordelivery via a catheter, stent, wire, or other intraluminal device.Delivery via such devices may be especially useful for delivery to thebladder, urethra, ureter, rectum, or intestine.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

The compounds described herein can be alternatively administered byaerosol. This is accomplished by preparing an aqueous aerosol, liposomalpreparation, or solid particles containing the composition. A nonaqueous(e.g., fluorocarbon propellant) suspension could be used. Sonicnebulizers are preferred because they minimize exposing the agent toshear, which can result in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular composition,but typically include nonionic surfactants (Tweens, Pluronics, sorbitanesters, lecithin, Cremophors), pharmaceutically acceptable co-solventssuch as polyethylene glycol, innocuous proteins like serum albumin,oleic acid, amino acids such as glycine, buffers, salts, sugars, orsugar alcohols. Aerosols generally are prepared from isotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the active compound in theproper medium. Absorption enhancers can also be used to increase theflux of the compound across the skin. The rate of such flux can becontrolled by either providing a rate controlling membrane or dispersingthe compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.Exemplary ophthalmic formulations are described in U.S. Publication Nos.2005/0080056, 2005/0059744, 2005/0031697 and 2005/004074 and U.S. Pat.No. 6,583,124, the contents of which are incorporated herein byreference. If desired, liquid ophthalmic formulations have propertiessimilar to that of lacrimal fluids, aqueous humor or vitreous humor orare compatable with such fluids. A preferred route of administration islocal administration (e.g., topical administration, such as eye drops,or administration via an implant).

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.Pharmaceutical compositions suitable for parenteral administrationcomprise one or more active compounds in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a ligand, drug, or other materialother than directly into the central nervous system, such that it entersthe patient's system and thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsulated matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

The preparations of agents may be given orally, parenterally, topically,or rectally. They are, of course, given by forms suitable for eachadministration route. For example, they are administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, infusion; topically by lotion or ointment; and rectally bysuppositories. Oral administration is preferred.

For use in the methods of this invention, active compounds can be givenper se or as a pharmaceutical composition containing, for example, 0.1to 99.5% (more preferably, 0.5 to 90%) of active ingredient incombination with a pharmaceutically acceptable carrier.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a compound at a particular targetsite.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally, and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds, whichmay be used in a suitable hydrated form, and/or the pharmaceuticalcompositions of the present invention, are formulated intopharmaceutically acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient that is effective to achieve the desired therapeutic responsefor a particular patient, composition, and mode of administration,without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound or combination ofcompounds employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound(s) being employed, the duration of the treatment,other drugs, compounds and/or materials used in combination with theparticular compound(s) employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts. In general, thecompositions of this invention may be provided in an aqueous solutioncontaining about 0.1-30% w/v of a compound disclosed herein, among othersubstances, for parenteral administration. Typical dose ranges are fromabout 0.01 to about 50 mg/kg of body weight per day, given in 1 singleor 2-4 divided doses. Each divided dose may contain the same ordifferent compounds of the invention.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the therapeutically effective amount of thepharmaceutical composition required. For example, the physician orveterinarian could start doses of the pharmaceutical composition orcompound at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. A “therapeutically effective amount” of acompound with respect to the subject method of treatment, refers to anamount of the compound(s) in a preparation which, when administered aspart of a desired dosage regimen (to a mammal, preferably a human)alleviates a symptom, ameliorates a condition, or slows the onset ofdisease conditions according to clinically acceptable standards for thedisorder or condition to be treated or the cosmetic purpose, e.g., at areasonable benefit/risk ratio applicable to any medical treatment. It isgenerally understood that the effective amount of the compound will varyaccording to the weight, sex, age, and medical history of the subject.Other factors which influence the effective amount may include, but arenot limited to, the severity of the patient's condition, the disorderbeing treated, the stability of the compound, and, if desired, anothertype of therapeutic agent being administered with the compound of theinvention. A larger total dose can be delivered by multipleadministrations of the agent. Methods to determine efficacy and dosageare known to those skilled in the art (Isselbacher et al. (1996)Harrison's Principles of Internal Medicine 13 ed., 1814-1882, hereinincorporated by reference).

In general, a suitable daily dose of an active compound used in thecompositions and methods of the invention will be that amount of thecompound that is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above.

If desired, the effective daily dose of the active compound may beadministered as one, two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In certain embodiments of the presentinvention, the active compound may be administered two or three timesdaily. In preferred embodiments, the active compound will beadministered once daily.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

In certain embodiments, compounds of the invention may be used alone orconjointly administered with another type of therapeutic agent. As usedherein, the phrase “conjoint administration” refers to any form ofadministration of two or more different therapeutic compounds such thatthe second compound is administered while the previously administeredtherapeutic compound is still effective in the body (e.g., the twocompounds are simultaneously effective in the patient, which may includesynergistic effects of the two compounds). For example, the differenttherapeutic compounds can be administered either in the same formulationor in a separate formulation, either concomitantly or sequentially. Incertain embodiments, the different therapeutic compounds can beadministered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72hours, or a week of one another. Thus, an individual who receives suchtreatment can benefit from a combined effect of different therapeuticcompounds.

This invention includes the use of pharmaceutically acceptable salts ofcompounds of the invention in the compositions and methods of thepresent invention. In certain embodiments, contemplated salts of theinvention include, but are not limited to, alkyl, dialkyl, trialkyl ortetra-alkyl ammonium salts. In certain embodiments, contemplated saltsof the invention include, but are not limited to, L-arginine,benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol,diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine,ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium,L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine,potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine,tromethamine, and zinc salts. In certain embodiments, contemplated saltsof the invention include, but are not limited to, Na, Ca, K, Mg, Zn orother metal salts.

The pharmaceutically acceptable acid addition salts can also exist asvarious solvates, such as with water, methanol, ethanol,dimethylformamide, dimethylsulfoxide, and the like. Mixtures of suchsolvates can also be prepared. The source of such solvate can be fromthe solvent of crystallization, inherent in the solvent of preparationor crystallization, or adventitious to such solvent. In someembodiments, a solvate of a disclosed compound can be adimethylsulfoxide solvate.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1)water-soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3)metal-chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

EXAMPLES Analytical Methods for Examples 1-5 X-Ray Powder Diffraction

X-Ray Powder Diffraction patterns were collected on a Bruker D8diffractometer using Cu Kα radiation (40 kV, 40 mA), 0-20 goniometer,and divergence of V4 and receiving slits, a Ge monochromator and aLynxeye detector. The instrument is performance checked using acertified Corundum standard (NIST 1976). The software used for datacollection was Diffrac Plus XRD Commander v2.6.1 and the data wereanalysed and presented using Diffrac Plus EVA v15.0.0.0.

Samples were run under ambient conditions as flat plate specimens usingpowder as received. The sample was gently packed into a cavity cut intopolished, zero-background (510) silicon wafer. The sample was rotated inits own plane during analysis. The details of the data collection are:

-   -   Angular range: 2 to 42° 2θ    -   Step size: 0.05° 2θ    -   Collection time: 0.5 s/step

HPLC

Purity analysis was performed on an Agilent HP1100 series systemequipped with a diode array detector and using ChemStation softwarevB.04.03 using the method detailed below in Table 1.

TABLE 1 HPLC Parameters Parameter Value Type of method Reversed phasewith gradient elution Sample Preparation 1 mg/mL in aqueous 20 mMammonium acetate, pH 5.0 Column Waters Atlantis C18, 100 Å, 3 μM ColumnTemperature 25° C. Injection Volume 5 μL Detector Wavelength, 254 nm, 4nm Bandwidth Flow Rate 1.0 mL/min Mobile Phase A 20 mM aqueous ammoniumacetate, pH 5.0 Mobile Phase B methanol Time (min) % Mobile Phase AGradient Timetable 0 100 5 5 8 5 8.1 100 16.1 100

Polarised Light Microscopy (PLM)

Samples were studied on a Leica LM/DM polarised light microscope with adigital video camera for image capture. A small amount of each samplewas placed on a glass slide, 15 mounted in immersion oil and coveredwith a glass slip, the individual particles being separated as well aspossible. The sample was viewed with appropriate magnification andpartially polarised light, coupled to a λ false-color filter.

Example 1: Synthesis of Form 1 by Crystallisation from Methanol

Amorphous nicotinamide mononucleotide (565 mg) was weighed into a glassvial and methanol (10.0 mL) added. The resulting slurry was stirred atroom temperature for 4 h and then a further portion of MeOH (10.0 mL)added. The white solid present was isolated by filtration and then driedunder vacuum at room temperature for ca. 16 h to give crystalline betanicotinamide mononucleotide Form 1, as shown by XRPD analysis. (459 mg,81% recovery).

Example 2: Synthesis of Form 1 by Crystallisation from Water

Amorphous beta nicotinamide mononucleotide (605 mg) was weighed into aglass vial and deionised water (600 μL) added. After brief vortexing, aclear solution was formed. A portion of this solution (ca. 200 μL) wasdispensed into a separate vial and cooled to 5° C. for ca. 16 h. Anevolved white solid was isolated by filtration and shown by XRPDanalysis to be crystalline beta nicotinamide mononucleotide Form 1(yield not determined).

Example 3: Synthesis of Form 1 by Vapor Diffusion for SCXRD

Amorphous beta nicotinamide mononucleotide (605 mg) was weighed into aglass vial and deionised water (600 μL) added. After brief vortexing, aclear solution was formed. A portion of this solution (100 μL) wasdispensed into a separate vial, which itself was placed into a largervial containing methanol (500 μL) such that vapor can diffuse freelybetween both vials. The larger vial was sealed and stored at RT for ca.16 h, after which time a white solid had evolved. This solid was sampledand shown by SCXRD to be crystalline beta nicotinamide mononucleotideForm 1.

Example 4: Synthesis of Form 2

Amorphous beta nicotinamide mononucleotide (568 mg) was weighed into aglass vial and DMSO (10.0 mL) added. The resulting slurry was stirred atroom temperature for ca. 20 h. The white solid present was then isolatedby filtration, washed with acetone (3×1 mL) and dried under vacuum atroom temperature for ca. 16 h to give crystalline beta nicotinamide

mononucleotide Form 2 (501 mg, 72% recovery*). * based on assumptionthat material is a DMSO mono-solvate

Example 5: Three Month Stability and Forced Degradation Study forAmorphous and Crystalline NMN

Samples of amorphous NMN and NMN crystalline Form 1 were stored assolids at 25° C./0% Relative Humidity (RH), and 40° C./75% RH for threemonths. The samples were prepared in duplicate with an offset of 2weeks. Each replicate was stored in a different container.

Replicate 1 was charged in an open HPLC vial that was placed in a sealedscintillation vial containing a saturated solution of the relevantinorganic salt (Table 2). These samples were analysed by HPLC, 1H NMR,XRPD and Polarized Light Microscopy (PLM) at 4, 8, and 12 week timepoints.

Replicate 2 was stored in sealed box with a recipient containing asaturated solution of the relevant inorganic salt (Table 2). Thesesamples were analysed by HPLC at 2, 6, 10 and 12 week time points.Samples at 25° C./0% RH were stored in a sealed box containing adesiccant agent (P₂O₅).

TABLE 2 Inorganic Salt/ Conditions Dessicant Agent 25° C./0% RH P₂O₅ 25°C./60% RH NH₄NO₃ 25° C./97% RH K₂SO₄ 40° C./75% RH NaCl

Amorphous material remained unchanged in terms of solid form andparticle morphology after storage for 12 weeks at 25° C., 0% RH. A dropin purity was observed from 98.2% (time 0) to 95.5% (time 12 weeks). Themain growing impurity observed by HPLC eluted at RRT 1.73 andcorresponded to nicotinamide (2.8% at the 12 weeks' time point). (SeeTables 3 and 4.)

TABLE 3 HPLC Purity Profiles of Amorphous NMN and Crystal Form 1 NMN att = 0 J07086 RME-1304-063-01 (Amorphous) (Crystalline) RRT Area % RRTArea % 0.70 0.47 0.69 0.15 0.76 0.10 0.75 0.09 1.00 98.56  1.00 99.52 —— 1.31 0.06 — — 1.46 0.01 — — 1.48 0.03 — — 1.53 0.01 1.76 0.87 1.740.12

The amorphous material crystallized when stored under 25% or greaterrelative humidity, giving Form 1 with acicular morphology. Chemicalpurity was shown to decrease upon storage at all the tested conditions,especially at 40° C./75% RH. The main growing impurity observed was alsonicotinamide (RRT=1.73). Purity of the material after 12 weeks at 40°C./75% RH was determined as 67.5% (replicate 2) and the nicotinamideabundance was 20.6%.

Crystalline Form 1 remained unchanged in terms of solid form andparticle morphology at all conditions tested. The material remainedchemically pure (˜99.3%) after 4 weeks of storage at all the testedconditions. A slight drop in purity was observed beyond that time point.Storage at 25° C./0% RH proved to be the most favorable conditions forsample stability as the analysis after 12 weeks showed the sample to be98.9% pure and 0.38% nicotinamide. The largest change in purity wasobserved for the samples stored at high temperature (40° C.) or highhumidity (75% RH, 97% RH). Purity dropped from 99.5% to 95.8% after 12weeks at 25°/97% RH and to 90.7% after 12 weeks at 40° C./75% RH. Themost abundant impurity at all conditions was nicotinamide (RRT=1.73min). This impurity increased from 0.12% (time 0) to 3.46% after 12weeks at both, 40° C./75% RH and 25° C./97% RH.

The HPLC results were consistent among replicates, however, fasterdegradation was observed with Replicate 1. A potential influence mayhave been the smaller size of the containers which required less time toequilibrate to the corresponding storage conditions. Tables 4-7 provideresults obtained for Replicates 1 and 2.

TABLE 4 Stability and Forced Degradation Study: Results for AmorphousMaterial Replicate 1 Known Time Purity Impurity* Sample ID ConditionsPoint Observations XRPD (%) (%) PLM J07087_25_0_4w 25° C., 4 weeks whitesolid Unchanged 96.9 1.81 Unchanged J07087_25_0_8w 0% RH 8 weeks whitesolid Unchanged 95.8 2.44 Unchanged J07087_25_0_12w 12 weeks  whitesolid Unchanged 95.0 2.81 Unchanged J07087_25_60_4w 25° C., 4 weeksbrown solid Form 1 96.9 2.13 acicular 60% RH particles up to ~75-100 μmJ07087_25_60_8w 8 weeks brown solid, Form 1 95.2 3.46 — compactedJ07087_25_60_12w 12 weeks  brown solid, Form 1 95.7 3.46 acicularcompacted particles up to ~75-100 μm J07087_25_97_4w 25° C., 4 weekswhite solid, very Form 1 97.1 2.44 acicular 97% RH wet particles up to~75-100 μm J07087_25_97_8w 8 weeks partially n/a — — — deliquesced,yellow liquid J07087_25_97_12w 12 weeks  deliquesced, n/a — — — yellowliquid J07087_40_75_4w 40° C., 4 weeks brown compacted Form 1 91.3 6.70acicular 75% RH solid particles up to ~75-100 μm J07087_40_75_8w 8 weeksbrown compacted Form 1 76.9 14.23  — solid J07087_40_75_12w 12 weeks black solid, very Form 1 — — — wet^($) *nicotinamide (RRT = 1.73 min)^($)HPLC analysis not performed as weight could not be stabilised forsample preparation. ¹H NMR spectra were consistent with the structure ofthe materials of all samples.

TABLE 5 Stability and Forced Degradation Study: Results for CrystallineMaterial Replicate 1 Known Time Purity Impurity * Sample ID ConditionsPoint Observations XRPD (%) (%) PLM RME-1304-63- 25° C., 4 weeks whitesolid Unchanged 99.4 0.25 acicular 01_25_0_4w 0% RH particles up to ~75μm RME-1304-63- 8 weeks white solid Unchanged 99.2 0.33 acicular01_25_0_8w particles up to ~75 μm RME-1304-63- 12 weeks  white solidUnchanged 98.9 0.38 acicular 01_25_0_12w particles up to ~75 μmRME-1304-63- 25° C., 4 weeks light brown solid Unchanged 99.3 0.34acicular 01_25_60_4w 60% RH particles up to ~75 μm RME-1304-63- 8 weekslight brown solid, Unchanged 98.9 0.63 acicular 01_25_60_8w looseparticles particles up to ~75 μm RME-1304-63- 12 weeks  light brownsolid, Unchanged 98.2 0.98 acicular 01_25_60_12w loose particlesparticles up to ~75 μm RME-1304-63- 25° C., 4 weeks white solidUnchanged 99.3 0.49 acicular 01_25_97_4w 97% RH particles <75 μmRME-1304-63- 8 weeks white solid Unchanged 98.3 1.38 acicular01_25_97_8w particles up to 75- 100 μm RME-1304-63- 12 weeks  whitesolid, wet Unchanged 95.8^($) 3.46 acicular 01_25_97_12w particles <75μm RME-1304-63- 40° C., 4 weeks white solid Unchanged 99.2 0.46 acicular01_25_75_4w 75% RH particles up to ~75 μm RME-1304-63- 8 weeks whitesolid with Unchanged 97.7 1.38 acicular 01_25_75_8w orange spotsparticles up to ~75 μm RME-1304-63- 12 weeks  brown solid, veryUnchanged 90.7 3.46 acicular 01_25_75_12w wet particles <75 μm *nicotinamide (RRT = 1.73 min) ^($)Weight could not be stabilised.Approximate value was taken. ¹HNMR spectra were consistent with thestructure of the materials of all samples.

TABLE 6 Forced Degradation Study Results for Amorphous MaterialReplicate 2 Known Time Purity Impurity* Sample ID Conditions PointObservations (%) (%) J07087_25_60_2w 25° C.,  2 weeks light yellowsolid, 97.4 2.14 60% RH very compacted J07087_25_60_6w  6 weeks lightbrown, 97.4 2.06 compacted J07087_25_60_10w 10 weeks light brown, 96.02.92 compacted J07087_R2_25_60_12w 12 weeks light brown, 96.2 2.32compacted J07087_25_97_2w 25° C.,  2 weeks white compacted 97.8 1.79 97%RH solid J07087_25_97_6w  6 weeks white solid, very 96.5 3.01 wetJ07087_25_97_10w 10 weeks white solid, very 94.9 4.39 wetJ07087_25_97_12w 12 weeks white solid, very 91.5 7.02 wetJ07087_40_75_2w 40° C.,  2 weeks brown compacted 94.6 4.61 75% RH solidJ07087_40_75_6w  6 weeks dark brown solid 88.0 8.22 J07087_40_75_10w 10weeks black solid 75.9 16.18 J07087_R2_40_75_12w 12 weeks black solid,very wet 67.5^($) 20.60 *nicotinamide (RRT = 1.73 min) ^($)Weight couldnot be stabilised. Approximate value was taken.

TABLE 7 Forced Degradation Study Results for Amorphous MaterialReplicate 2 Known Sample ID Time Purity Impurity* RME-1304-63-01-_(—)Conditions Point Observations (%) (%) _25_60_2w 25° C.,  2 weeks whitesolid 99.6 0.19 _25_60_6w 60% RH  6 weeks light brown 99.5 0.21compacted solid _25_60_10w 10 weeks light brown 99.1 0.35 compactedsolid R2_25_60_12w 12 weeks light brown 98.8 0.23 compacted solid_25_97_2w 25° C.,  2 weeks white solid 99.5 0.22 _25_97_6w 97% RH  6weeks white solid, 99.4 0.38 very wet _25_97_10w 10 weeks white solid,wet 98.6 0.77 R2_25_97_12w 12 weeks white solid, wet 98.4 0.55 _40_75_2w40° C.,  2 weeks white solid 99.5 0.22 _40_75_6w 75% RH  6 weeks whitesolid 99.4 0.27 with orange spots _40_75_10w 10 weeks light brown 98.80.49 loose particles R2_40_75_12w 12 weeks light brown 97.4 0.77 looseparticles *nicotinamide (RRT = 1.73 min)

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

1. A crystalline Form 1 of a compound having the structure of formula (I),


2. (canceled)
 3. The crystalline form of claim 1, having 2θ values 20.03; 20.14; 21.83; and 25.73.
 4. The crystalline form of claim 3, having 2θ values 20.03; 20.14; 21.03; 21.83; 23.08; 23.39; 25.73; and 26.59.
 5. The crystalline form of claim 4, having 2θ values 7.70; 11.54; 12.64; 16.03; 18.99; 20.03; 20.14; 20.83; 21.03; 21.83; 23.08; 23.39; 25.48; 25.73; 26.59; and 29.78.
 6. The crystalline form of claim 5, having 2θ values 7.70; 9.95; 11.54; 12.64; 16.03; 18.18; 18.99; 19.16; 19.44; 20.03; 20.14; 20.83; 21.03; 21.83; 22.44; 23.08; 23.39; 23.89; 24.08; 24.53; 24.68; 25.05; 25.48; 25.73; 26.08; 26.59; 27.33; 27.67; 29.78; and 29.92.
 7. The crystalline compound of claim 6, having an XRD pattern substantially as shown in FIG.
 1. 8-13. (canceled)
 14. A pharmaceutical composition comprising the crystalline Form 1 of claim 1 and one or more pharmaceutically acceptable excipients.
 15. A method for preparing a crystalline Form 1 of a compound having the structure of formula (I):

comprising: a) providing a mixture of a compound of formula (I) in a solvent; and b) crystallizing the compound of formula (I) from the mixture comprising the compound of formula (I). 16-18. (canceled)
 19. The method of claim 15, wherein the solvent comprises water.
 20. (canceled)
 21. The method of claim 15, wherein the mixture comprising the compound of formula (I) is a solution, and the step of crystallizing the compound of formula (I) from the mixture comprises bringing the solution to supersaturation to cause the compound of formula (I) to precipitate out of solution.
 22. The method of claim 21, wherein the step of bringing the solution to supersaturation comprises slowly adding an anti-solvent, allowing the solution to cool, reducing the volume of the solution, or any combination thereof.
 23. The method of claim 21, wherein the step of bringing the solution to supersaturation comprises cooling the solution to ambient temperature or lower.
 24. The method of claim 15, wherein the mixture comprising the compound of formula (I) is a slurry.
 25. The method of claim 15, further comprising isolating the crystalline form of the compound.
 26. The method of claim 25, wherein isolating the crystalline compound comprises filtering the crystalline form of the compound from the mixture.
 27. The method of claim 25, further comprising drying the crystalline form of the compound under reduced pressure.
 28. (canceled) 